Cervical cancer is the second most common cancer diagnosis in women and is linked to high-risk human papillomavirus infection 99.7% of the time. Currently, 12,000 new cases of invasive cervical cancer are diagnosed in US women annually, resulting in 5,000 deaths each year. Furthermore, there are approximately 400,000 cases of cervical cancer and close to 200,000 deaths annually worldwide. Human papillomaviruses (HPVs) are one of the most common causes of sexually transmitted disease in the world. Overall, 50-75% of sexually active men and women acquire genital HPV infections at some point in their lives. An estimated 5.5 million people become infected with HPV each year in the US alone, and at least 20 million are currently infected. The more than 100 different isolates of HPV have been broadly subdivided into high-risk and low-risk subtypes based on their association with cervical carcinomas or with benign cervical lesions or dysplasias.
HPVs are a diverse group of relative small DNA viruses and are the etiologic agents of epithelial outgrowths, or papillomas. The DNAs of papilloma virus can be divided into the Early Region Genes (E5, E6, E7) and Late Region Gene (L1, L2) where E regions are postulated to encode proteins needed for replication and transformation while the L regions to encode the viral capsid proteins. These proteins related to HPV are biomarkers used to detect HPV in samples. In humans, specific papillomavirus types have been detected in, and associated with over 99% of cervical cancer biopsies. These are considered the high-risk types and include, in orders of prevalence, HPV types-16, 18, 31, 33, 35, 45, 51, 52, and 56.
A number of lines of evidence point to HPV infections as the etiological agents of cervical cancers. Multiple studies in the 1980's reported the presence of HPV variants in cervical dysplasias, cancer, and in cell lines derived from cervical cancer. Further research demonstrated that the E6-E7 region of the genome from oncogenic HPV 18 is selectively retained in cervical cancer cells, suggesting that HPV infection could be causative and that continued expression of the E6-E7 region is required for maintenance of the immortalized or cancerous state. The following year, Sedman et al. demonstrated that the E6-E7 genes from HPV 16 were sufficient to immortalize human keratinocytes in culture. Barbosa et al. demonstrated that although E6-E7 genes from high risk HPVs could transform cell lines, the E6-E7 regions from low risk, or non-oncogenic variants such as HPV 6 and HPV 11 were unable to transform human keratinocytes. More recently, Pillai et al. examined HPV 16 and 18 infection by in situ hybridization and E6 protein expression by immunocytochemistry in 623 cervical tissue samples at various stages of tumor progression and found a significant correlation between histological abnormality and HPV infection.
Current treatment paradigms are focused on the actual cervical dysplasia rather than the underlying infection with HPV. Women are screened by physicians annually for cervical dysplasia and are treated with superficial ablative techniques, including cryosurgery, laser ablation and excision. As the disease progresses, treatment options become more aggressive, including partial or radical hysterectomy, radiation or chemotherapy. A significant unmet need exists for early and accurate diagnosis of oncogenic HPV infection as well as for treatments directed at the causative HPV infection, preventing the development of cervical cancer by intervening earlier in disease progression. Human papillomaviruses characterized to date are associated with lesions confined to the epithelial layers of skin, or oral, pharyngeal, respiratory, and, most importantly, anogenital mucosae. Specific human papillomavirus types, including HPV 6 and 11, frequently cause benign mucosal lesions, whereas other types such as HPV 16, 18, and a host of other strains, are predominantly found in high-grade lesions and cancer. Individual types of human papillomaviruses (HPV) which infect mucosal surfaces have been implicated as the causative agents for carcinomas of the cervix, anus, penis, larynx and the buccal cavity, occasional periungal carcinomas, as well as benign anogenital warts. The identification of particular HPV types is used for identifying patients with premalignant lesions who are at risk of progression to malignancy. Although visible anogenital lesions are present in some persons infected with human papillomavirus, the majority of individuals with HPV genital tract infection do not have clinically apparent disease, but analysis of cytomorphological traits present in cervical smears can be used to detect HPV infection.
At the present time, the primary methodology for public health screening for cervical cancer has been the annual Pap smear test. The main problems associated with the Pap smear test have been the high rate of false negatives. Nucleic acid assays developed for HPV screening offer much better sensitivity and specificity. They include Souther blotting, Dot blot, Filter in situ Hybridization (FISH), In situ hybridization, Hybrid capture and Polymerase chain reaction (PCR). Among the nucleic acid assays, Digene's Hybrid capture assay and Roche's PCR based assay hold most of the market. However, both assays tend to suffer from high cost, sampling difficulties and contamination issues. In addition, the oncogenicity of HPV has been shown to be protein based. As such, detection of HPV DNA or RNA may lead to unnecessary medical procedures that the body's immune system may solve naturally.
Recently, immunoassays have been developed for HPV diagnostic application. E6-Associated Protein (E6-AP), a polypeptide that stably associates with E6 in the presence or absence of p53 has been isolated and proposed to be used to detect the presence of “high risk” human papillomaviruses (U.S. Pat. No. 5,914,384). A broad antibody based sandwich assay for detection of HPV protein, particularly HPV-E6 or HPV-E7 was described in U.S. Pat. No. 6,214,541 B1. U.S. Pat. No. 4,777,239 describes a series of 17 synthetic peptides which are said to be capable of raising antibodies to HPV-16 and thus may be useful for diagnostic purpose. EU Patent 0 412 762 describes polypeptides which are antagonists of biochemical interaction of HPV E7 protein and the retinoblastoma gene protein, and which are said to be useful in the treatment of genital warts and cervical cancer. U.S. Patent Application Publication No. US 2003/0064477 A1 describes a putative tumor suppressor protein designated E6-TP1 (for E6-targeted protein) that can act as a therapeutic agent for treatment or prevention of HPV-associated carcinomas. U.S. Patent Application Publication No. US 2004/0110925 A1 describes an isolated protein sequence or peptide from HPV for detecting or diagnosing cancer or cellular abnormalities, said isolated proteins sequence or peptide selected from the group consisting of: E2, E6, E7, and E8 regions of HPV type 16, 18, 31, 33, 35, 45, 51, 52, 56, and 58. U.S. Patent Application Publication No. US 2004/0018487 A1 describes a method of detecting the presence of an oncogenic HPV E6 protein in a sample by capturing an oncogenic HPV E6 protein with a PDZ domain polypeptide. Each of these publications and all publications mentioned herein are incorporated in their entirety by reference herein.
Traditional assay methods also fail to provide a single test to detect all (or even more than one) of the different types of biomarkers which are associated with HPV. Most of the immunoassays presently used, for example, can only detect the native E6 or E7 proteins of human papillomavirus types 16, 18, 31, 33, 35, 45, 51, 52, or 56, which make the sampling procedure more difficult. Detection of E6 protein using antibodies is difficult because E6 that is made in a human cells contains a number of structural modifications, e.g., disulfide bonds and phosphate groups, that cause wild-type E6 protein made in bacterial systems, or chemically synthesized E6 peptides, to not recognize E6 protein in human cells. Thus, one would have to run many separate assays to have any chance of a thorough detection technique and yet still risk a detection error.
Traditional assay methods also fail to detect more than one type of protein in a single testing. Immunoassays, for example, require multiple runs of a protein array, without cross-reactions.
It would therefore be advantageous to have a new diagnostic tool for the detection of HPV in a patient that provides a single test to detect more than one or all of the different biomarkers which are associated with HPV, higher sensitivity and/or specificity for the detection of HPV than other methods, a lower false-positive rate of diagnosis, and/or a reduction in the number of patients requiring further screening. It would also be advantageous to use the capabilities of SELDI-MS to detect and identify biomarkers capable of correctly classifying samples as those originating from patients having HPV.